PCSK9 inhibition attenuates alcohol-associated neuronal oxidative stress and cellular injury.

Alcohol Use Disorder (AUD) is a persistent condition linked to neuroinflammation, neuronal oxidative stress, and neurodegenerative processes. While the inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has demonstrated effectiveness in reducing liver inflammation associated with alcohol, its impact on the brain remains largely unexplored. This study aimed to assess the effects of alirocumab, a monoclonal antibody targeting PCSK9 to lower systemic low-density lipoprotein cholesterol (LDL-C), on central nervous system (CNS) pathology in a rat model of chronic alcohol exposure. Alirocumab (50 mg/kg) or vehicle was administered weekly for six weeks in 32 male rats subjected to a 35 % ethanol liquid diet or a control liquid diet (n = 8 per group). The study evaluated PCSK9 expression, LDL receptor (LDLR) expression, oxidative stress, and neuroinflammatory markers in brain tissues. Chronic ethanol exposure increased PCSK9 expression in the brain, while alirocumab treatment significantly upregulated neuronal LDLR and reduced oxidative stress in neurons and brain vasculature (3-NT, p22phox). Alirocumab also mitigated ethanol-induced microglia recruitment in the cortex and hippocampus (Iba1). Additionally, alirocumab decreased the expression of pro-inflammatory cytokines and chemokines (TNF, CCL2, CXCL3) in whole brain tissue and attenuated the upregulation of adhesion molecules in brain vasculature (ICAM1, VCAM1, eSelectin). This study presents novel evidence that alirocumab diminishes oxidative stress and modifies neuroimmune interactions in the brain elicited by chronic ethanol exposure. Further investigation is needed to elucidate the mechanisms by which PCSK9 signaling influences the brain in the context of chronic ethanol exposure.

µ-Opioid receptor antagonism facilitates the anxiolytic-like effect of oxytocin in mice.

Mood and anxiety disorders are leading causes of disability worldwide and are major contributors to the global burden of diseases. Neuropeptides, such as oxytocin and opioid peptides, are important for emotion regulation. Previous studies have demonstrated that oxytocin reduced depression- and anxiety-like behavior in male and female mice, and opioid receptor activation reduced depression-like behavior. However, it remains unclear whether the endogenous opioid system interacts with the oxytocin system to facilitate emotion regulation in male and female mice. We hypothesized that opioid receptor blockade would inhibit the anxiolytic- and antidepressant-like effects of oxytocin. In this study, we systemically administered naloxone, a preferential µ-opioid receptor antagonist, and then intracerebroventricularly administered oxytocin. We then tested mice on the elevated zero maze and the tail suspension tests, respective tests of anxiety- and depression-like behavior. Contrary to our initial hypothesis, naloxone potentiated the anxiolytic-like, but not the antidepressant-like, effect of oxytocin. Using a selective µ-opioid receptor antagonist, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, and a selective κ-opioid receptor antagonist, norbinaltorphimine, we demonstrate that µ-opioid receptor blockade potentiated the anxiolytic-like effect of oxytocin, whereas κ-opioid receptor blockade inhibited the oxytocin-induced anxiolytic-like effects. The present results suggest that endogenous opioids can regulate the oxytocin system to modulate anxiety-like behavior. Potential clinical implications of these findings are discussed.

Alcohol Use Disorder Treatment: Problems and Solutions.

Alcohol use disorder (AUD) afflicts over 29 million individuals and causes more than 140,000 deaths annually in the United States. A heuristic framework for AUD includes a three-stage cycle-binge/intoxication, withdrawal/negative affect, and preoccupation/anticipation-that provides a starting point for exploring the heterogeneity of AUD with regard to treatment. Effective behavioral health treatments and US Food and Drug Administration-approved medications are available but greatly underutilized, creating a major treatment gap. This review outlines challenges that face the alcohol field in closing this treatment gap and offers solutions, including broadening end points for the approval of medications for the treatment of AUD; increasing the uptake of screening, brief intervention, and referral to treatment; addressing stigma; implementing a heuristic definition of recovery; engaging early treatment; and educating health-care professionals and the public about challenges that are associated with alcohol misuse. Additionally, this review focuses on broadening potential targets for the development of medications for AUD by utilizing the three-stage heuristic model of addiction that outlines domains of dysfunction in AUD and the mediating neurobiology of AUD.

No time to lose: the current state of research in rapid-acting psychotherapeutics.

The vast majority of treatments for psychiatric and substance use disorders take weeks to work. Notable exceptions to this rule exist, with some treatments such as intravenous ketamine resolving symptoms in minutes to hours. Current research is focused on identifying novel approaches to rapid-acting psychotherapeutics. Promising results from studies of novel classes of drugs and innovative brain stimulation therapies are currently being studied through both clinical and pre-clinical research, as described here. Research focused on understanding neurobiological mechanisms, effective therapeutic context, and implementation approaches are needed to maximize the potential reach of these therapies.

Cold nociception as a measure of hyperalgesia during spontaneous heroin withdrawal in mice.

Opioids are powerful analgesic drugs that are used clinically to treat pain. However, chronic opioid use causes compensatory neuroadaptations that result in greater pain sensitivity during withdrawal, known as opioid withdrawal-induced hyperalgesia (OWIH). Cold nociception tests are commonly used in humans, but preclinical studies often use mechanical and heat stimuli to measure OWIH. Thus, further characterization of cold nociception stimuli is needed in preclinical models. We assessed three cold nociception tests-thermal gradient ring (5-30 °C, 5-50 °C, 15-40 °C, and 25-50 °C), dynamic cold plate (4 °C to -1 °C at -1 °C/min, -1 °C to 4 °C at +1 °C/min), and stable cold plate (10 °C, 6 °C, and 2 °C)-to measure hyperalgesia in a mouse protocol of heroin dependence. On the thermal gradient ring, mice in the heroin withdrawal group preferred warmer temperatures, and the results depended on the ring's temperature range. On the dynamic cold plate, heroin withdrawal increased the number of nociceptive responses, with a temperature ramp from 4 °C to -1 °C yielding the largest response. On the stable cold plate, heroin withdrawal increased the number of nociceptive responses, and a plate temperature of 2 °C yielded the most significant increase in responses. Among the three tests, the stable cold plate elicited the most robust change in behavior between heroin-dependent and nondependent mice and had the highest throughput. To pharmacologically characterize the stable cold plate test, we used µ-opioid and non-opioid receptor-targeting drugs that have been previously shown to reverse OWIH in mechanical and heat nociception assays. The full µ-opioid receptor agonist methadone and µ-opioid receptor partial agonist buprenorphine decreased OWIH, whereas the preferential µ-opioid receptor antagonist naltrexone increased OWIH. Two N-methyl-d-aspartate receptor antagonists (ketamine, MK-801), a corticotropin-releasing factor 1 receptor antagonist (R121919), a ß2-adrenergic receptor antagonist (butoxamine), an α2-adrenergic receptor agonist (lofexidine), and a 5-hydroxytryptamine-3 receptor antagonist (ondansetron) had no effect on OWIH. These data demonstrate that the stable cold plate at 2 °C yields a robust, reliable, and concise measure of OWIH that is sensitive to opioid agonists.

Sex and hormonal status influence the anxiolytic-like effect of oxytocin in mice.

Anxiety and depression are highly prevalent psychiatric disorders, affecting approximately 18% of the United States population. Evidence indicates that central oxytocin mediates social cognition, social bonding, and social anxiety. Although it is well-established that oxytocin ameliorates social deficits, less is known about the therapeutic effects of oxytocin in non-social contexts. We hypothesized that positive effects of oxytocin in social contexts are attributable to intrinsic effects of oxytocin on neural systems that are related to emotion regulation. The present study investigated the effect of intracerebroventricular (ICV) oxytocin administration (i.e., central action) on anxiety- and depression-like behavior in C57Bl/6J mice using non-social tests. Male and female mice received an ICV infusion of vehicle or oxytocin (100, 200, or 500 ng), then were tested in the elevated zero maze (for anxiety-like behavior) and the tail suspension test (for depression-like behavior). Oxytocin dose-dependently increased open zone occupancy and entries in the elevated zero maze and reduced immobility duration in the tail suspension test in both sexes. Oxytocin decreased anxiety and depression-like behavior in male and female mice. The observed effect of oxytocin on anxiolytic-like behavior appeared to be driven by the males. Given the smaller anxiolytic-like effect of oxytocin in the female mice and the established interaction between oxytocin and reproductive hormones (estrogen and progesterone), we also explored whether oxytocin sensitivity in females varies across estrous cycle phases and in ovariectomized females that were or were not supplemented with estrogen or progesterone. Oxytocin reduced anxiety-like behavior in female mice in proestrus/estrus, ovariectomized females (supplemented or not with estrogen or progesterone), but not females in metestrus/diestrus. Additionally, oxytocin reduced depression-like behavior in all groups tested with slight differences across the various hormonal statuses. These results suggest that the effect of oxytocin in depression- and anxiety-like behavior in mice can be influenced by sex and hormonal status.

Theoretical Frameworks and Mechanistic Aspects of Alcohol Addiction: Alcohol Addiction as a Reward Deficit/Stress Surfeit Disorder.

Alcohol use disorder (AUD) can be defined by a compulsion to seek and take alcohol, the loss of control in limiting intake, and the emergence of a negative emotional state when access to alcohol is prevented. Alcohol use disorder impacts multiple motivational mechanisms and can be conceptualized as a disorder that includes a progression from impulsivity (positive reinforcement) to compulsivity (negative reinforcement). Compulsive drug seeking that is associated with AUD can be derived from multiple neuroadaptations, but the thesis argued herein is that a key component involves the construct of negative reinforcement. Negative reinforcement is defined as drug taking that alleviates a negative emotional state. The negative emotional state that drives such negative reinforcement is hypothesized to derive from the dysregulation of specific neurochemical elements that are involved in reward and stress within basal forebrain structures that involve the ventral striatum and extended amygdala, respectively. Specific neurochemical elements in these structures include decreases in reward neurotransmission (e.g., decreases in dopamine and opioid peptide function in the ventral striatum) and the recruitment of brain stress systems (e.g., corticotropin-releasing factor [CRF]) in the extended amygdala, which contributes to hyperkatifeia and greater alcohol intake that is associated with dependence. Glucocorticoids and mineralocorticoids may play a role in sensitizing the extended amygdala CRF system. Other components of brain stress systems in the extended amygdala that may contribute to the negative motivational state of withdrawal include norepinephrine in the bed nucleus of the stria terminalis, dynorphin in the nucleus accumbens, hypocretin and vasopressin in the central nucleus of the amygdala, and neuroimmune modulation. Decreases in the activity of neuropeptide Y, nociception, endocannabinoids, and oxytocin in the extended amygdala may also contribute to hyperkatifeia that is associated with alcohol withdrawal. Such dysregulation of emotional processing may also significantly contribute to pain that is associated with alcohol withdrawal and negative urgency (i.e., impulsivity that is associated with hyperkatifeia during hyperkatifeia). Thus, an overactive brain stress response system is hypothesized to be activated by acute excessive drug intake, to be sensitized during repeated withdrawal, to persist into protracted abstinence, and to contribute to the compulsivity of AUD. The combination of the loss of reward function and recruitment of brain stress systems provides a powerful neurochemical basis for a negative emotional state that is responsible for the negative reinforcement that at least partially drives the compulsivity of AUD.

Pharmacological GHSR (ghrelin receptor) blockade reduces alcohol binge-like drinking in male and female mice.

Ghrelin is a peptide that is produced by endocrine cells that are primarily localized in the stomach. Ghrelin receptors (GHSR) are expressed in the brain and periphery. Preclinical and clinical studies support a role for ghrelin in alcohol drinking and seeking. The GHSR has been suggested to be a potential pharmacotherapeutic target for alcohol use disorder (AUD). However, the role of the ghrelin system and its potential modulation by biological sex on binge-like drinking has not been comprehensively investigated. The present study tested six GHSR antagonists in an alcohol binge-like drinking procedure in male and female mice. Systemic administration of the GHSR antagonists JMV2959, PF-5190457, PF-6870961, and HM-04 reduced alcohol intake in both male and female mice. YIL-781 decreased intake in males, and LEAP2 (likely peripherally restricted) did not reduce intake in mice of either sex. We also administered LEAP2 and JMV2959 intracerebroventricularly to investigate whether the effects of GHSR blockade on alcohol intake are mediated by central receptors. The central administration of LEAP2 and JMV2959 decreased alcohol intake, particularly in high-drinking animals. Finally, in a preliminary experiment, an anti-ghrelin vaccine was examined for its potential effect on binge-like drinking and had no effect. In all experiments, there was a lack of meaningful sex differences. These findings suggest that central GHSR mediates binge-like alcohol intake. These data reveal novel pharmacological compounds with translational potential in the treatment of AUD and provide further evidence of the GHSR as a potential treatment target for AUD.

The glucagon-like peptide-1 (GLP-1) analogue semaglutide reduces alcohol drinking and modulates central GABA neurotransmission.

Growing evidence indicates that the glucagon-like peptide-1 (GLP-1) system is involved in the neurobiology of addictive behaviors, and GLP-1 analogues may be used for the treatment of alcohol use disorder (AUD). Here, we examined the effects of semaglutide, a long-acting GLP-1 analogue, on biobehavioral correlates of alcohol use in rodents. A drinking-in-the-dark procedure was used to test the effects of semaglutide on binge-like drinking in male and female mice. We also tested the effects of semaglutide on binge-like and dependence-induced alcohol drinking in male and female rats, as well as acute effects of semaglutide on spontaneous inhibitory postsynaptic currents (sIPSCs) from central amygdala (CeA) and infralimbic cortex (ILC) neurons. Semaglutide dose-dependently reduced binge-like alcohol drinking in mice; a similar effect was observed on the intake of other caloric/noncaloric solutions. Semaglutide also reduced binge-like and dependence-induced alcohol drinking in rats. Semaglutide increased sIPSC frequency in CeA and ILC neurons from alcohol-naive rats, suggesting enhanced GABA release, but had no overall effect on GABA transmission in alcohol-dependent rats. In conclusion, the GLP-1 analogue semaglutide decreased alcohol intake across different drinking models and species and modulated central GABA neurotransmission, providing support for clinical testing of semaglutide as a potentially novel pharmacotherapy for AUD.

Heroin- and Fentanyl-Induced Respiratory Depression in a Rat Plethysmography Model: Potency, Tolerance, and Sex Differences.

The opioid overdose death toll in the United States is an ongoing public health crisis. We characterized the magnitude and duration of respiratory depression, the leading cause of death in opioid overdose cases, induced by heroin or fentanyl and the development of tolerance in male and female rats. We used whole-body plethysmography to first establish dose-response curves by recording breathing for 60 minutes post-intravenous opioid injection. We then tested the development of respiratory tolerance to acute heroin or fentanyl over several weeks and to chronic fentanyl with acute fentanyl or heroin challenge. Heroin and fentanyl each provoked dose-dependent respiratory depression. Heroin caused prolonged (45-60 minute) respiratory depression in female and male rats, characterized by decreased frequency, tidal volume, and minute ventilation and increased inspiratory time and apneic pause. Fentanyl produced similar changes with a shorter duration (10-15 minutes). High-dose heroin or fentanyl produced robust respiratory depression that was slightly more severe in females and, when given intermittently (acute doses 2 to 3 weeks apart), did not lead to tolerance. In contrast, chronic fentanyl delivered with an osmotic minipump resulted in tolerance to acute fentanyl and heroin, characterized by a shorter duration of respiratory depression. This effect persisted during withdrawal in males only. Our model and experimental design will allow for investigation of the neurobiology of opioid-induced respiratory depression and for testing potential therapeutics to reverse respiratory depression or stimulate breathing. SIGNIFICANCE STATEMENT: Fentanyl was more potent and had shorter duration in producing respiratory depression than heroin in both sexes, whereas female rats were more sensitive than males to heroin-induced respiratory depression. Tolerance/cross-tolerance develops in chronic fentanyl administration but is minimized with long interadministration intervals.

Extended access to fentanyl vapor self-administration leads to addiction-like behaviors in mice: Blood chemokine/cytokine levels as potential biomarkers.

Rodent models are useful for understanding the mechanisms that underlie opioid addiction, but most preclinical studies have focused on rewarding and consummatory aspects of opioids without components of dependence-induced escalation of drug taking or seeking. We characterized several opioid-related behaviors in mice using a model of vaporized fentanyl self-administration. Male and female C57BL/6J mice were assigned to short-access (ShA; 1 h, nondependent) or long-access (LgA; 6 h, dependent) fentanyl vapor self-administration and subsequently tested in a battery of behavioral tests, followed by blood collection during withdrawal. Compared with mice in the ShA group, mice in the LgA group escalated their fentanyl intake, were more motivated to work to obtain the drug, exhibited greater hyperalgesia, and exhibited greater signs of naloxone-precipitated withdrawal. Principal component analysis indicated the emergence of two independent behavioral constructs: "intake/motivation" and "hyperalgesia/punished seeking." In mice in the LgA condition only, "hyperalgesia/punished seeking" was associated with plasma levels of proinflammatory interleukin-17 (IL-17), chemokine (C-C motif) ligand 4 (CCL-4), and tumor necrosis factor α (TNF-α). Overall, the results suggest that extended access to opioids leads to addiction-like behavior, and some constructs that are associated with addiction-like behavior may be associated with levels of the proinflammatory cytokines/chemokines IL-17, TNF-α, and CCL-4 in blood.

Initial Pharmacological Characterization of a Major Hydroxy Metabolite of PF-5190457: Inverse Agonist Activity of PF-6870961 at the Ghrelin Receptor.

Preclinical and clinical studies have identified the ghrelin receptor [growth hormone secretagogue receptor (GHSR)1a] as a potential target for treating alcohol use disorder. A recent phase 1a clinical trial of a GHSR1a antagonist/inverse agonist, PF-5190457, in individuals with heavy alcohol drinking identified a previously undetected major hydroxy metabolite of PF-5190457, namely PF-6870961. Here, we further characterized PF-6870961 by screening for off-target interactions in a high-throughput screen and determined its in vitro pharmacodynamic profile at GHSR1a through binding and concentration-response assays. Moreover, we determined whether the metabolite demonstrated an in vivo effect by assessing effects on food intake in male and female rats. We found that PF-6870961 had no off-target interactions and demonstrated both binding affinity and inverse agonist activity at GHSR1a. In comparison with its parent compound, PF-5190457, the metabolite PF-6870961 had lower binding affinity and potency at inhibiting GHSR1a-induced inositol phosphate accumulation. However, PF-6870961 had increased inhibitory potency at GHSR1a-induced ß-arrestin recruitment relative to its parent compound. Intraperitoneal injection of PF-6870961 suppressed food intake under conditions of both food restriction and with ad libitum access to food in male and female rats, demonstrating in vivo activity. The effects of PF-6870961 on food intake were abolished in male and female rats knockout for GHSR, thus demonstrating that its effects on food intake are in fact mediated by the GHSR receptor. Our findings indicate that the newly discovered major hydroxy metabolite of PF-5190457 may contribute to the overall activity of PF-5190457 by demonstrating inhibitory activity at GHSR1a. SIGNIFICANCE STATEMENT: Antagonists or inverse agonists of the growth hormone secretagogue receptor (GHSR)1a have demonstrated substantial potential as therapeutics for alcohol use disorder. We here expand understanding of the pharmacology of one such GHSR1a inverse agonist, PF-5190457, by studying the safety and pharmacodynamics of its major hydroxy metabolite, PF-6870961. Our data demonstrate biased inverse agonism of PF-6870961 at GHSR1a and provide new structure-activity relationship insight into GHSR1a inverse agonism.

Alcohol and aging - An area of increasing concern.

Alcohol use is increasing among adults 65 and older and the size of this population is expanding rapidly. Aging is associated with systemic inflammation, sleep disturbances, cancers, cognitive decline, and increased risk of injury and death from falls and other accidents. Alcohol misuse exacerbates and accelerates these age-related changes. Older drinkers are more sensitive to acute alcohol-induced impairments in memory, coordination, reaction time, and driving performance. Oxidative stress and DNA damage resulting from chronic heavy alcohol consumption contribute to an increased risk of cancer, liver disease, and cardiovascular disease. Medication use increases with age and many medications prescribed to older adults can interact negatively with alcohol. The rapid expansion of the population aged 65 and older, combined with higher levels of alcohol use and AUD in the Baby Boomer cohort than the preceding generation, could significantly increase the burden of alcohol on the healthcare system resulting from AUD and alcohol-related injuries and diseases. Screening and brief intervention for hazardous alcohol use among older patients along with education regarding potential interactions between alcohol and medications could substantially reduce the risk of harms from alcohol but currently is underutilized.

Spironolactone as a potential new pharmacotherapy for alcohol use disorder: convergent evidence from rodent and human studies.

Evidence suggests that spironolactone, a nonselective mineralocorticoid receptor (MR) antagonist, modulates alcohol seeking and consumption. Therefore, spironolactone may represent a novel pharmacotherapy for alcohol use disorder (AUD). In this study, we tested the effects of spironolactone in a mouse model of alcohol drinking (drinking-in-the-dark) and in a rat model of alcohol dependence (vapor exposure). We also investigated the association between spironolactone receipt for at least 60 continuous days and change in self-reported alcohol consumption, using the Alcohol Use Disorders Identification Test-Consumption (AUDIT-C), in a pharmacoepidemiologic cohort study in the largest integrated healthcare system in the US. Spironolactone dose-dependently reduced the intake of sweetened or unsweetened alcohol solutions in male and female mice. No effects of spironolactone were observed on drinking of a sweet solution without alcohol, food or water intake, motor coordination, alcohol-induced ataxia, or blood alcohol levels. Spironolactone dose-dependently reduced operant alcohol self-administration in dependent and nondependent male and female rats. In humans, a greater reduction in alcohol consumption was observed among those who received spironolactone, compared to propensity score-matched individuals who did not receive spironolactone. The largest effects were among those who reported hazardous/heavy episodic alcohol consumption at baseline (AUDIT-C ≥ 8) and those exposed to ≥ 50 mg/day of spironolactone. These convergent findings across rodent and human studies demonstrate that spironolactone reduces alcohol use and support the hypothesis that this medication may be further studied as a novel pharmacotherapy for AUD.